Autoimmune disorders are conditions caused by an immune response against the body's own tissues. Autoimmune disorders result in destruction of one or more types of body tissues, abnormal growth of an organ, or changes in organ function. The disorder may affect only one organ or tissue type or may affect multiple organs and tissues.
Type 1 autoimmune diabetes (T1D) in humans and the NOD mouse model is a polygenic T-cell dependent autoimmune disease, characterized by the selective destruction of the β cells of the islets of Langerhans (1-3) arising from a breakdown in tolerance to β cell antigens (4) in susceptible individuals who have inherent defects in critical immunomodulatory mechanisms (5) (e.g., individuals who exhibit a pathogenic rather than a protective immune response to self). Pancreatic exocrine and endocrine cells that share the same developmental lineage as the β-cell and are often in direct contact with β-cells are largely unaffected, and insulitis resolves once the β-cells are lost, which suggests that the autoimmune targets are largely β-cell specific. Many of the known diabetes autoantigens, such as insulin and IGRP reflect this cell specificity, however, the list of known immune targets is by no means comprehensive. This applies especially to the antigens recognized by the cellular arm of the immune system, and the targets of the majority of CD4+ diabetogenic T-cells remain elusive in spite of more than a decade of investigation. Few new targets have emerged in recent years using the immunological screening procedures that originally identified such molecules as GAD65, IA-2 and ICA 69.
Many individuals with a strong genetic predisposition to T1D never develop overt disease (50% of monozygotic twins with one diabetic member remain discordant) and non-germ line encoded factors significantly influence the rate of progression of T1D (7). This low penetrance and variable natural history points to the importance of environmental factors, and also the stochastic nature of the immune response itself. Therapeutic strategies that shift this balance towards a more toleragenic environment have the potential to prevent or slow the progression of autoimmunity to destructive insulitis.
There appears to be a restricted number of islet-cell reactive T-cell clones in early pancreatic infiltrates in NOD mice (16; 17) and disease progression appears to involve epitope spreading and (18) avidity maturation of early T-cell responses (19) along with the recruitment of new autoantigens (20). Numerous islet-specific CD4+ and CD8+ T-cell clones have been isolated from spleen, lymph nodes or islet infiltrates of pre- or newly diabetic NOD mice (21-25) whose cognate antigens are poorly defined and do not appear to correspond to any of the known serological markers of diabetes autoimmunity in humans such as GAD65, the insulin granule membrane proteins ICA512 (IA-2) and phogrin (IA-2β) (20), carboxypeptidase E (26), ICA69 (27) and sulphated glycolipids (28). The molecular targets of autoreactive T-cells may include either an unidentified component of β-cells, or known β-cell proteins that are intrinsically unable to elicit a humoral response. Accordingly, defining the cognate antigens for them remains an important goal, and with the recent advances in genomic and proteomic techniques, now appears a realistic aim. Thus the target of the well studied NY8.3 CD8+ clone (23) was recently shown to be a peptide derived from the β-cell protein IGRP (islet glucose 6-phosphatase related protein) by using a combination of a sensitive bioassay and analysis of proteomic peptides eluted from H-2Kd molecules from NIT1 insulinoma cells (29).
Although the humoral response per se probably contributes little to the pathogenesis of the disease (30), identification of the molecular targets of B-lymphocytes is also an important objective, since B-cells play a role in antigen presentation in T1D (31). In addition, circulating autoantibodies provide useful pre-clinical markers for diabetic autoimmunity. The production of high affinity antibodies is a T-dependent process, and thus it is reasonable to expect that molecules recognized by autoantibodies should also be the targets of autoreactive diabetogenic T-cells. This hypothesis appears correct, at least for insulin, phogrin and GAD65 (32-34). The list of known targets, while long, is far from comprehensive. This is highlighted by the fact that serological diagnosis of pre-T1D determined by immunohistochemistry of human pancreas is still the most sensitive index, simply because it defines additional targets that are not among the autoantigens defined in molecular terms.
There is a continuing debate about whether there are primary or initiating autoantigens in T1D, and in other autoimmune diseases. For TID, the best candidate in the NOD mouse at present is insulin (33; 35-38) although it has also been proposed that IGRP can play this crucial role (29; 39; 40) based upon the precursor frequency of T-cells recognizing this antigen in islet infiltrates. An alternative view is that disease results from polyclonal activation due to a breakdown in normal toleragenic mechanisms that would otherwise generate regulatory cells directed to the same molecules (41). Given the latter scenario, the inventors hypothesize that any molecule that is a significant target of autoimmunity in T1D is a candidate for use in antigen-based therapy. Effective tolerization strategies in NOD mice based on immunization with the native epitopes of insulin (42; 43), GAD65 (44-48), HSP 65 (49) and IGRP (50) appear to bear this out. Moreover, the inventors' preliminary studies with IA-2 and phogrin show that a known mouse and human peptide epitope (peptide 7 (51)) likewise slows the emergence of disease when administered neonatally to NOD mice. Other known T-cell targets, including IAPP (52), IMOGEN 38 (53-57) appear not to have been characterized in this regard.
Examples of putative molecular mimicry between autoantigens and viral proteins based upon sequence homology abound in the scientific literature (58). For example, in the case of T1D, cross-reactivity between GAD65 and Coxsackie B3 P2-C protein (59) or human cytomegalovirus major DNA-binding protein (60), and IA-2 with rotavirus VP7 (61) or Coxsackie B4 VP1 (62) have been proposed. Similarly, mimicry between proinsulin and GAD65 has also been postulated (63). Exposure to a molecular mimic could conceivably either trigger autoimmunity, or protect against it, by establishing and consolidating immune networks. Epidemiological studies of autoimmune triggering by infectious agents in man have not been particularly informative possibly because of the long prodrome of the disease and failure to identify a specific organism (or rare serotype of a common pathogen) that is involved (14). Protective responses that are central to the “hygiene hypothesis” by their very nature are more difficult to establish.
The non-obese diabetic (NOD) mouse is currently the best model of human T1D (8) where three stages of disease progression are evident, namely, expansion of autoreactive T-cells (“checkpoint 0”), their homing to the pancreatic islets (“checkpoint 1”), and the transition from a benign peri-insulitis to an invasive insulitis resulting in β-cell destruction (“checkpoint 2”) (9). It has been postulated that passage through “checkpoint 1” coincides with a wave of islet cell apoptosis that leads to enhanced presentation of β-cell antigens in the pancreatic lymph nodes. A related hypothesis suggests that exposure to novel antigens in the gut at this time (when weaning is occurring) results in activation of Th1 polarized T-cells that subsequently migrate to the pancreas and perturb the response in the draining nodes such that an immunogenic response to islet cell antigens results. A similar involvement of post-natal islet cell apoptosis (10), congenital β-cell abnormalities (11), and dietary (12; 13) or enteroviral (14) triggers have also been proposed in human T1D, although at present, their relative roles (if any), and generality as casual factors, remain controversial. Nevertheless it is clear that islet cell antigens are critical to the disease process (15), and that a detailed knowledge of their molecular characteristics is essential both to the rational design of immunotherapies, and in the monitoring and identification of at-risk individuals.
There are upwards of 2 million type 1 diabetes patients in the United States alone, all of whom require lifelong multiple daily doses of insulin to stay alive. The majority of these individuals will suffer from the complications of diabetes within their lifetime and it is estimated that at any age, their lifespan may be reduced by up to a third. Prevention or even slowing of the development of the disease would thus have immense social and economic benefits. There are a number of experimental approaches to diabetes prevention and reversal that have proven to be very effective in rodent models of T1D and which are currently being tested in clinical trials. For example, preliminary findings with anti-CD3 monoclonal antibody treatment are particularly promising. To be efficacious, any immune-based therapy requires diagnostic tests to establish whether intervention is appropriate and when to implement therapy relative to staging of the disease. Once therapy is initiated, it is essential to assess its short term and long term efficacy. Diagnostic assays based on humoral or cellular autoreactivity to autoantigens that are useful in monitoring disease progression are essential in making any decision to treat a patient and in the subsequent monitoring of treatment outcome.
Therefore, there is a continued need in the art for improved diagnostic assays for T1D, as well as new immunotherapeutics based on disease targets. In addition, many targets identified in one autoimmune disease, such as TID, will also be targets in other autoimmune diseases, thereby expanding the available diagnostic and therapeutic approaches in a variety of autoimmune conditions.